(1) Field of the Invention
The present invention relates to heat-stable and protease-resistant antibacterial activity in excretory-secretory products (ESP) of Trichuris suis. The antibacterial activity is not more than 10,000 MW; is resistant to boiling, trypsin, and pronase E; has a bactericidal mode of action; and is effective against Gram positive and Gram negative bacteria, including Escherichia coli, Campylobacter jejuni, Campylobacter coli, and Staphylococcus aureus. The antibacterial activity is useful in applications for killing or inhibiting the growth of microorganisms, in particular bacteria.
(2) Description of Related Art
Compounds with antibacterial activity have been identified from a wide array of invertebrates, including parasitic nematodes (Wardlaw et al., J. Appl. Bacteriol. 76: 36–41 (1994); Kato, Zoo. Sci. 12: 225–30 (1995)). These factors constitute a primitive humoral defense system. It is not surprising that metazoan parasites inhabiting the gastrointestinal tract (GI) produce antibacterial substances, since they are in a microbe-rich environment containing potential pathogens. For example, a potent antibacterial activity was found in the body fluid of Ascaris suum, a nematode parasitizing the pig small intestine (Wardlaw et al., J. Appl. Bacteriol. 76: 36–41 (1994); Kato, Zoo. Sci. 12: 225–30 (1995)). The bactericidal activity was heat stable and less than 14,000 MW in size. Subsequently, three humoral defense activities (antibacterial, bacteriolytic, and agglutinating) were detected in the body fluid of A. suum (Kato, Zoo. Sci. 12: 225–30 (1995)).
The A. suum antibacterial factor (referred to as ASABF) has been well-characterized (Kato and Komatsu, J. Biol. Chem. 271: 30493–30498 (1996)). ASABF is a heat-stable and trypsin-sensitive peptide of 71 amino acids (Kato and Komatsu, J. Biol. Chem. 271: 30493–30498 (1996)). ASABF has structural and functional similarities to the defensins of insects/arthropods. Both are cysteine-rich, cationic peptides that are more effective against Gram positive bacteria than Gram negative bacteria (Kato and Komatsu, J. Biol. Chem. 271: 30493–30498 (1996)). ASABF has significant sequence identity with the proteins deduced from a cDNA sequence (yk150c7) and from a putative gene (T22H6.5) of Caenorhabditis elegans, a free-living nematode (Kato and Komatsu, J. Biol. Chem. 271: 30493–30498 (1996)).
A gene family of saposin-like proteins has been identified in C. elegans, with one of them (T07C4.4) having antibacterial activity when expressed as a recombinant in E. coli (Banyai and Patthy, Biochim. Biophys. Acta 1429: 259–64 (1998)). The putative products of these C. elegans genes are similar to the amoebapores of Entamoeba histolytica and a putative amoebapore-related protein of the liver fluke Fasciola hepatica in that they consist of a single saposin-like domain and a secretory signal peptide (Banyai and Patthy, Biochim. Biophys. Acta 1429: 259–64 (1998)). Amoebapores of E. histolytica, an invasive protozoan pathogen, are pore-forming peptides with antibacterial an cytolytic activities, which function by formation of ion channels in target cell membranes (Andra, et al., FEBS Letters 385: 96–100 (1996); Leippe et al., Molec. Microbiol. 14: 895–904 (1994)).
Antibacterial activity in invertebrates is quite common. Antibacterial peptides have been described from the silkworm, Bombyx mori (Chowdhury, et al., Biochem. Biophys. Res. Comm. 214: 271–8 (1995); Hara and Yamakawa, J. Biol. Chem. 270: 29923–7, (1995); Kim et al., Biochem. Biophys. Res. Comm. 246: 388–92 (1998)). A proline-rich antibacterial peptide from the earthworm, Lumbricus rubellus, has been reported (Cho et al., Biochim. Biophys. Acta 1408: 67–76 (1998)). Antibacterial agents have also been detected in two other annelid species, Nereis diversicolor (Salzet-Raveillon et al., Cell. Molec. Biol. 39: 105–14 (1993)) and Eisenia foetida (Kauschke and Mohrig, Devel. Compar. Immunol. 11: 331–41 (1987)).
Most of the antimicrobial agents identified from invertebrates are peptides that exhibit structural similarities. Insect defensins are cationic, cysteine-rich peptides forming intra-molecular disulfide bridges that appear in the hemolymph after bacterial challenge or injury (Cociancich et al., J. Biol. Chem. 268: 19239–45 (1993)). They have potent antibacterial activity against Gram positive bacteria mediated by disruption of the permeability barrier of the cytoplasmic membrane (Cociancich et al., J. Biol. Chem. 268: 19239–45 (1993)). The myticins found in the haemocytes of the mussel, Mytilus galloprovincialis, are cysteine-rich and exemplify antibacterial peptides originating as precursors with signal sequences that require proteolytic events to activate the mature peptide (Mitta et al., Eur. J. Biochem. 265: 71–8 (1999)). Antibacterial peptides offer a means for treating or preventing bacterial infections that is not based upon an antibiotic mode of activity.
Antibiotics are commonly used to prevent bacterial infections. However, because many antibiotics have been used with relative abandon over the past half century, many microorganisms have developed resistance to many of the antibiotics. As a consequence, many antibiotics have been rendered ineffective at preventing bacterial infections. Unless new antibiotics or alternative antibacterial treatments are developed, the world will be staring at a public health crisis of immense proportions. Therefore, there is a dire need for novel antibacterial compositions for preventing bacterial infections.